Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated herein by reference as though set forth in full.
Insects and other lower eukaryotes, such as nematodes and plants, occupy an interesting evolutionary niche in glycobiology because they produce N-glycoproteins, but they typically process their N-linked glycans less extensively than mammals (1,2). This difference between lower and higher eukaryotic protein N-glycosylation pathways is biotechnologically significant because insects and plants are used to produce recombinant mammalian glycoproteins for many different biomedical research applications (3-7). Insect and mammalian protein N-glycosylation pathways each begin with the co-translational transfer of N-glycan precursors to nascent proteins (1,8). These precursors are subsequently trimmed and elongated by enzymes localized in the endoplasmic reticulum and Golgi apparatus of insect and mammalian cells to produce a common intermediate with the structure Manα6(GlcNAcβ2Manα3)Manβ4 GlcNAcβ4GlcNAc-R. In mammalian cells, this intermediate is elongated by various glycosyltransferases to produce complex N-glycans, which often have terminal sialic acid residues. In contrast, insect cells usually fail to elongate this same intermediate and convert it, instead, to paucimannose N-glycans with the core structure Manα6(Manα3)Manβ4GlcNAcβ4GlcNAc-R. An unusual β-N-acetylglucosaminidase is responsible for the production of these structures (9). This enzyme specifically removes the terminal N-acetylglucosamine residue from the α3 branch of Manα6 (GlcNAcβ2Manα3)Manβ4GlcNAcβ4GlcNAc-R, simultaneously eliminating the intermediate required for N-glycan elongation and producing the core paucimannose glycan typically found on insect cell-derived N-glycoproteins. This same enzyme is also responsible for the production of core paucimannose N-glycans in nematodes (10,11) and plants (12,13). Thus, the presence of a processing β-N-acetylglucosaminidase is a key difference between the protein N-glycosylation pathways of lower and higher eukaryotes.
In the seminal insect study on this topic, Altmann and coworkers (9) demonstrated that IPLB-Sf21AE, a cell line derived from the lepidopteran insect S. frugiperda (14), has a membrane-associated β-N-acetylglucosaminidase activity that can specifically cleave the terminal N-acetylglucosamine residue from the α3 branch of a biantennary N-glycan in vitro. Subsequently, it was shown that cell lines derived from E. acrea, another lepidopteran insect, produced hybrid and complex N-glycans containing terminal N-acetylglucosamine or galactose residues because they lack this intracellular β-N-acetylglucosaminidase activity (15). Together, these studies strongly supported the idea that the N-glycosylation pathway of at least some insect cells includes a processing β-N-acetylglucosaminidase, as described above. However, unequivocal proof of this concept awaited the isolation of an insect gene encoding this enzyme, together with evidence that the gene product had the substrate specificity of the N-glycan processing enzyme.
The first proof of this kind was provided by a more recent study from Altmann's group, in which they demonstrated that the D. melanogaster fused lobes (Dm-fdl) gene encodes the specific, processing β-N-acetylglucosaminidase in this organism (16). Importantly, this study demonstrated that the Dm-fdl gene product has several features distinguishing it from degradative hexosaminidases and chitinases, which also have β-N-acetylglucosaminidase activities. These features included its specificity for the terminal N-acetylglucosamine residue linked to the α3 branch of N-glycan substrates and its inability to degrade chito-oligosaccharides. Furthermore, it was shown that flies lacking a functional fdl gene produced a higher proportion of N-glycans with terminal N-acetylglucosamine residues linked to the α3 branch than wild type. These findings, together with the finding that the D. melanogaster hexosaminidase genes (hexo1 and hexo-2) encode enzymes that can cleave chito-oligosaccharides, but not N-glycans, strongly suggested that Dm-FDL is the β-N-acetylglucosaminidase responsible for N-glycan processing in this fly. These properties also were consistent with the idea that Dm-FDL is an ortholog of the lepidopteran insect N-glycan processing enzyme first detected by Altmann and coworkers (1995) in microsomal membranes from IPLB-Sf21AE cells.
Subsequently, two lab groups independently reported molecular cloning of genes encoding β-N-acetylglucosaminidases from Sf9 cells, which are a clonal derivative of the IPLB-Sf21AE cell line (17,18). Our group described the isolation of three β-N-acetylglucosaminidase genes from Sf9 cells, which were designated SfGlcNAcase-1, -2, and -3 (18). SfGlcNAcase-1 was clearly distinct from the other two, which were nearly identical to each other and appeared to be allelic variants of the same gene. Further analysis of the SfGlcNAcase-1 and SfGlcNAcase-3 gene products showed that they had high sequence homology to known hexosaminidases and that each also had β-N-acetylglucosaminidase activity when assayed against relevant substrates. However, neither had the tight α3 branch specificity of the processing enzyme activity originally described by Altmann and coworkers (1995). In fact, each could remove the terminal N-acetylglucosamine residues from either the α3 or the α6 branch of various N-glycan substrates and each also was able to release N-acetylglucosamine monomers from a chito-oligosaccharide substrate. Accordingly, we concluded that none of these S. frugiperda genes encoded the N-glycan processing enzyme, but rather, that they encoded broad-spectrum β-N-acetylglucosaminidases that are more likely to be involved in N-glycan and chitin degradation. In a similar study, Tomiya and coworkers (2006) also molecularly cloned two allelic variants of an Sf9 cell β-N-acetylglucosaminidase gene, which they termed Sfhex. Further analysis of the Sfhex gene product, which is identical to the gene product we designated SfGlcNAcase-3, confirmed that the SfGlcNAcase-3/Sfhex gene product lacks the α3 branch specificity of the processing enzyme activity originally described by Altmann and coworkers. However, because this enzyme had a 2- to 5-fold higher preference for the terminal N-acetylglucosamine residue on the α3 branch of an N-glycan substrate, Tomiya and coworkers (2006) concluded that the SfGlcNAcase-3/Sfhex gene encodes the processing β-N-acetylglucosaminidase of Sf9 cells.